The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE2 levels in aqueous humor. Treatment with inhibitors of NOS (NG-nitro-L-arginine methyl ester, L-NAME, 50 mg/kp i.p.) or COX (diclofenac, 30 micrograms, topically), alone or in combination, were compared to a saline-treated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE2 have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.

The ocular pharmacokinetics of topical diclofenac sodium was studied in two experimental models of ocular inflammation and compared to physiological conditions. Keratitis or uveitis were induced by intrastromal injection of clove oil or by intravitreal lipopolysaccharide in rabbits. The control eyes were not inflamed. Simultaneously to the induction of inflammation, 30 µl of 0.1% diclofenac were applied topically in the right eye. Diclofenac levels were measured by HPLC in the cornea, aqueous humor (AH), iris/ciliary body (ICB) and plasma 30 min, 1, 3, 6 and 12 h after application. In physiological conditions, diclofenac reached a peak level in the cornea and ICB at 30 min slowly decreasing afterwards. Low levels of diclofenac were found in AH. In keratitic eyes, two peak levels which were significantly higher than in the controls were found in the cornea 30 min and 3 h after application. Diclofenac concentrations in keratitic AH and ICB were lower than in controls. In uveitic eyes, corneal and ICB levels peaked at 30 min, being significantly higher than in controls, and decreased quickly to very low levels at 1 h after application. In uveitic AH, diclofenac levels were lower than in controls. Plasma levels were very low (less than 0.1 µg/ml) in all experimental groups. It is concluded that the ocular pharmacokinetics of topical diclofenac is affected by inflammatory processes in the eye, reaching higher levels in the target tissues.

Age-related macular degeneration and other eye diseases, such as diabetic retinopathy, are probably linked to the effects of oxygen radicals derived from light or metabolic reactions. We have investigated the effects of hypoxia on bovine retinal pigmented epithelial cells (RPE) and the response of these cells to two antioxidants that have previously shown a beneficial action against free radical-linked senescent involution. The main results of the study were as follows: (i) Hypoxia induced apoptotic damage on RPE cells, with LDH leakage and ATP reduction; (ii) both vitamin C (VC) and N-acetyl-cysteine (NAC) treatment protected against hypoxia-induced apoptosis, with less DNA fragmentation. In our opinion, these findings justify further experimental and clinical work to investigate the role of hypoxia in the mechanisms of age-related RPE injury and death as well as the potential of antioxidant administration to prevent or delay retinal degenerative processes caused by oxygen-dependent pathophysiological conditions.

Nitric oxide (NO) synthase inhibitors, such as N^G-nitro-L-arginine methyl ester (L-NAME), have been shown to attenuate endotoxin-induced uveitis (EIU) but they could increase leukocyte adhesion to the vascular endothelium. We hypothesize that a concomitant treatment with the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) in 50% dimethylsulf-oxide (DMSO, a hydroxyl radical scavenger) could improve the anti-inflammatory activity of L-NAME. EIU was induced in albino rabbits by intravitreal injection of 100 ng lipopolysaccharide. Animals were treated with multiple intraperitoneal injections of 50% DMSO in phosphate-buffered saline (PBS), NDGA (10 mg/kg) in 50% DMSO, L-NAME (50 mg/ kg) in PBS, or the combination NDGA+L-NAME. Uveitis was assessed by slit lamp examination, protein levels in aqueous humor, and myeloperoxidase (MPO) activity in the iris/ciliary body 6 h after induction. Nitrite, leukotriene B₄ (LTB₄), prostaglandin E₂ (PGE₂), platelet-activating factor (PAF) and interleukin-1β (IL·-1β) levels in aqueous humor were also determined. NDGA or L-NAME alone did not show a significant reduction of uveitis intensity, although a significant decrease in MPO or in proteins was found, respectively. The combination NDGA+L-NAME significantly reduced the uveitis intensity, MPO in the iris/ciliary body, and the levels of nitrites, LTB₄, PGE₂, and PAF in aqueous humor. IL-1β levels were lower than the detection limit of the radioimmunoassay in all treatment groups. We conclude that concomitant treatment with NDGA in DMSO improves the anti-inflammatory activity of L-NAME during the early phase of EIU, suggesting that the inhibition of NO synthesis could enhance leukocyte infiltration and the release of oxygen free radicals.

The aim of this work was to study the effect of various anti-inflammatory drugs on PGE₂ synthesis in cultured bovine corneal endothelial cells (BCECs) stimulated with calcium ionophore A₂₃₁₈₇ or lipopolysaccharide (LPS) of Salmonella typhimurium. NSAIDs were more potent in inhibiting LPS-stimulated PGE₂ synthesis. Diclofenac was more potent than indomethacin, although both drugs showed a 98% maximal inhibitory effect. Dexamethasone inhibited 80% of the A₂₃₁₈₇-stimulated PGE₂ synthesis and only 53% of the LPS-stimulated PGE₂ synthesis. Prednisolone did not show an inhibitory effect. The results demonstrate the inhibitory effect of NSAIDs and show differences between the activity of glucocorticoids on PGE₂ synthesis in BCECs. Prednisolone could not inhibit PGE₂ synthesis in these cells in our experimental conditions.